Interstitial cells of Cajal (ICC) are considered to be pacemaker cells in gastrointestinal tracts. ICC generate electrical rhythmicity (dihydropyridine-insensitive) as slow waves and drive spontaneous contraction of smooth muscles. Although cytosolic Ca 2؉ has been assumed to play a key role in pacemaking, Ca 2؉ movements in ICC have not yet been examined in detail. In the present study, using cultured cell clusters isolated from mouse small intestine, we demonstrated Ca 2؉ oscillations in ICC. Fluo-4 was loaded to the cell cluster, the relative amount of cytosolic Ca 2؉ was recorded, and ICC were identified by c-Kit immunoreactivity. We specifically detected Ca 2؉ oscillation in ICC in the presence of dihydropyridine, which abolishes Ca 2؉ oscillation in smooth muscles. The oscillation was coupled to the electrical activity corresponding to slow waves, and it depended on Ca 2؉ influx through a non-selective cation channel, which was SK&F 96365-sensitive and store-operated. We further demonstrated the presence of transient receptor potential-like channel 4 (TRP4) in caveolae of ICC. Taken together, the results infer that the Ca 2؉ oscillation in ICC is intimately linked to the pacemaker function and depends on Ca 2؉ influx mediated by TRP4. Interstitial cells of Cajal (ICC)1 are a distinct and unique cell population distributed in the gastrointestinal (GI) muscle layer of many vertebrates including humans (1-2). They are network-forming cells connected electrically with each other and with smooth muscle cells via gap junctions. GI muscle shows spontaneous rhythmical contractions accompanied by periodic electrical oscillation, i.e. so-called slow waves that are affected by neither tetradotoxin (TTX, blocker of nervous activity) nor dihydropyridine (blocker of L-type calcium channel) (3-5). It has been postulated that ICC are pacemaker cells that generate slow waves and induce spontaneous contractions of the smooth muscles.In the last decade, ICC were found to express the protooncogene c-kit and to develop depending upon activation of c-kit signal pathways (4, 6 -8). Expression of c-kit and/or c-Kit receptors has been used commonly as a marker of ICC in the GI muscle layers, and it also enables the isolation of ICC. Recent electrophysiological studies using isolated ICC have demonstrated periodic oscillations of the membrane current (9 -11). Thus, ICC have been considered as pacemaker cells in recent years.The intrinsic properties underlying the pacemaking mechanism in ICC have been emphasized in previous reports (11). Several groups have reported that generation of electrical rhythmicity involves Ca 2ϩ release through inositol trisphosphate (IP 3 ) type 1 receptor in the endoplasmic reticulum (ER) and subsequent Ca 2ϩ entry into mitochondria (12)(13)(14). transients in ICC and longitudinal smooth muscles. Their results suggested that [Ca 2ϩ ] i plays a crucial role in pacemaking and that Ca 2ϩ imaging at the tissue level is a useful technique to investigate slow wave propagation in GI muscle. It is, however, n...

Intracellular Ca2+ ([Ca2+]i) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca2+ release from intracellular Ca2+ stores produces [Ca2+]i oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca2+]i oscillations in ICCs are preserved in the presence of dihydropyridine Ca2+ antagonists, which suppress Ca2+ activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca2+]i oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca2+]i oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP3Rs. The reconstituted [Ca2+]i oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.

SUMMARY1. Whole-cell voltage clamp techniques were used to examine the properties of voltage-dependent Ca2" channel currents in single smooth muscle cells enzymatically dissociated from guinea-pig urinary bladder. Potassium currents were blocked with intracellular Cs'. A holding potential of -60 mV was normally applied.2. When the membrane potential was returned to the holding potential after a depolarizing step, tail currents were seen after depolarizations to positive potentials, and the size of the tail current increased with increasing positivity of the preceding depolarization.3. After depolarization to +80 mV (a potential at which little inward current flowed through the Ca2+ channels) tail currents on returning to the holding potential increased in size as the duration of the depolarization was increased. 4. Investigation of the mechanism mediating the tail currents showed that they were not flowing through non-selective cation channels, and had no contribution from Ca2+-activated C1-channels or Na+-Ca 2+ exchange.5. The tail currents and the inward currents evoked by a simple depolarizing test potential were equally decreased by nifedipine in a dose-dependent manner. This suggests that L-type Ca21 channels are responsible for both of the two types of inward currents. The inward currents were also inhibited in a similar manner when caffeine was applied.6. Although the tail currents evoked on stepping from + 80 mV to a holding potential of -60 mV increased in size with the duration of the conditioning potential, the total membrane Ca2+ conductance did not increase, since the inward currents evoked on stepping to + 20 mV (a potential at which the Ca2+ channels are still fully activated) did not change with time.7. The amplitude of the inward current evoked by a simple depolarizing test potential was similar to that evoked on stepping to the same test potential after preconditioning at + 80 mV, if the test potential was higher than + 20 mV. However, following repolarization to the holding potential, the amplitude of the subsequent tail current was larger and the deactivation time constant longer, after the conditioning depolarization. These results suggest that the voltage-dependent Ca2+

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This paper demonstrates the performance of magnetoimpedance (MI) sensors for biomagnetic field recording. The rms noise of an amorphous wire MI sensor with 600 turns of pick‐up coil was estimated as approximately 3 pT/Hz1/2 at 1 Hz. Here, we demonstrate that this sensitivity is enough to clearly detect magnetic cardiogram signals. We also measured the biomagnetic field around a smooth muscle tissue sample taken from a guinea‐pig. We discovered that the magnetic‐field waveform of a small tissue sample is similar to the voltage waveform measured by a microelectrode array in a smooth muscle sheet sample. The results suggest that the localized biomagnetic field generated by smooth muscle cells can be detected by an MI sensor. We concluded that the MI sensor is a promising device for use as a noninvasive tool to test cell activity or function through magnetic‐field measurement. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HT's actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca2+ and electric activities in ICC were performed by employing fluorescent Ca2+ imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca2+ antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca2+ oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT3 receptor antagonist suppressed spontaneous Ca2+ activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT3 receptor agonist, restored it. GR113808, a selective antagonist for 5-HT4, and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT3 receptors, had little effect on ICC Ca2+ activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT3 receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT3 receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome.

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster-a "gut"-from mouse embryonic stem (ES) cells (ES gut). Each ES gut

SUMMARY1. In the taenia isolated from the guinea-pig caecum, changes in free intracellular magnesium concentration ([Mg2+] condition. In addition to this, Ca2+ inhibits both Mg2+ efflux and influx, probably by reducing the membrane permeability to Mg2+ and by competing with Mg2+ in the Na+_Mg2+ exchange of the plasma membrane.